Determination of creatinine in human serum using isocratic HPLC with an aluminum oxide stationary phase.

An isocratic high-performance liquid chromatographic method that uses aluminum oxide as the stationary phase is developed for the quantitation of creatinine in human serum. Sample pretreatment includes ultrafiltration, and ultraviolet detection is performed at 240 nm. The within-day precision, expressed as a mean coefficient of variation (CV), is 0.8%; the total method CV is 1.2%. The inaccuracy of the method is +1.5, -0.3, and +0.5%, respectively, as determined with the National Institute of Standards and Technology standard reference materials 909, 909 a1, and 909 a2. The detection limit of the method is 1.6 pmol (180 pg). The absence of interferences is confirmed by chromatographically analyzing patient serums again after enzymatic breakdown of creatinine.

Four frequently used test systems for serum cholesterol evaluated by isotope dilution gas chromatography-mass spectrometry candidate reference method.

We evaluated the performance of four frequently used cholesterol test systems, using split-sample measurements with a panel of 79 patients' specimens and isotope dilution gas chromatography-mass spectrometry (ID GS-MS) as a comparison method. The test systems were from Beckman, a Boehringer Mannheim, Merck, and Johnson & Johnson Clinical Diagnostics, performed on the Synchron CX7, Hitachi 717, Mega, and Ektachem 250 analyzers, respectively. The liner regression data for the method comparison [ID GS-MS as independent variable (x)] were for Beckman: slope = 1.012, intercept = 0.0243 mmol/L, dispersion (S(y/x)) = 0.1303 mmol/l, and correlation coefficient (r) = 0.9867; for Boehringer Mannheim: slope = 1.002, intercept = 0.114 mmol/L, Sy/x = 0.0759 mmol/L, r = 0.9954; for Merck: slope = 1.034, intercept = -0.0613 mmol/L, Sy/x = 0.0886 mmol/L, r = 0.9941; and for Johnson & Johnson Clinical Diagnostics: slope = 1.007, intercept = 0.01 mmol/L, Sy/x = 0.15 mmol/L, and r = 0.9811. These data demonstrate excellent state-of-the-art cholesterol measurement for some of the most widely used test systems.

Candidate reference method for determining serum creatinine by isocratic HPLC: validation with isotope dilution gas chromatography-mass spectrometry and application for accuracy assessment of routine test kits.

We present a candidate Reference Method for determining creatinine in serum, based on isocratic HPLC. The chromatographic column, with alkaline-treated aluminum oxide as stationary phase, is eluted with an equivolume mixture of methanol/acetonitrile containing 70 mL/L aqueous NaOH (10 mmol/L), and ultraviolet absorbance is detected at 240 nm. We investigated the ruggedness of the method and validated its performance with isotope dilution gas chromatography-mass spectrometry (ID GC-MS) for a set of 22 patients' sera and 10 commercially available lyophilized control materials. The mean deviation from ID GC-MS was +0.1% (range, -2.2% to +2.2%). The between-day CV, calculated from six independent measurements performed on three different days, was 0.9% (range 0.2% to 1.9%); the within-run CV was 0.8%. The total error of the method was < 3%. These performance characteristics make the method suitable for target-setting of quality-control materials and accuracy assessment of routine test kits. For the latter application, done with split-sample measurements of a panel of 83 patients' specimens, we used the Boehringer Mannheim enzymatic creatinine PAP test on the Hitachi 911, the Kodak Ektachem single-slide enzymatic creatinine test on the Ektachem 700, the Merck Jaffé kinetic assay on the Mega analyzer, and the Roche Jaffé kinetic test on the Cobas Mira.

Evaluation of 2-iminoimidazolidin-4-one and thymine as respective internal standards for normal-phase and reversed-phase high-performance liquid chromatographic determination of creatinine in human serum.

We evaluated two internal standards for HPLC determination of creatinine in human serum after ultrafiltration: 2-iminoimidazolidin-4-one for normal-phase HPLC on aluminium oxide, and thymine for C18 reversed-phase HPLC. Detection of 2-iminoimidazolidin-4-one was done at the same wavelength as that used for creatinine, i.e. 240 nm. For thymine, the wavelength was switched to 280 nm. The suitability of the selected compounds to serve as an internal standard in the described measurement procedures, including ultrafiltration of serum, was evaluated from the precision and accuracy obtained. The method based on normal-phase HPLC with 2-iminoimidazolidin-4-one showed an imprecision expressed as R.S.D. ranging from 0.8 to 3.4% (mean: 2.1%) and an inaccuracy, calculated from the deviations from target values determined by isotope-dilution gas chromatography-mass spectrometry, ranging from -1.3 to +1.8% (mean: +0.4%). For the reversed-phase HPLC procedure with thymine, the imprecision ranged from 0.3 to 1.3% (mean: 1.0%) and the inaccuracy from +0.1 to +3.9% (mean: +1.7%). The occasional observation of interferences with 2-iminoimidazolidin-4-one limited the application of the normal-phase method to a certain extent.